This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. After insulin stimulation, the insulin receptor, a receptor tyrosine kinase, is downregulated by several mechanisms, including dephosphorylation by protein tyrosine phosphatases such as PTP1B and binding of the adapter protein Grb14. Grb14, a member of the Grb7/10/14 family, comprises an N-terminal poly-proline region, a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a BPS (between PH and SH2) region, and a C-terminal Src-homology-2 (SH2) domain. Our laboratory has previously shown that the BPS region of Grb14 directly inhibits the catalytic activity of the insulin receptor by binding as a pseudo-substrate in the active site of the kinase domain. This results in suppression of substrate phosphorylation and hence downregulation of insulin signaling. The C-terminal SH2 domain binds to the phosphorylated activation loop of the kinase to increase the affinity and specificity of the Grb14-insulin receptor interaction.Biochemical studies in our laboratory have shown that Grb14-mediated inhibition of insulin signaling also requires functional RA and PH domains. Our crystallographic studies showed that the two domains are structurally coupled. The RA and PH domains of Grb14 are thought to interact with the small GTPase Ras and phosphoinositides, respectively. We hypothesize that Ras activation serves as a timing mechanism for the negative-feedback inhibition of insulin signaling by Grb14. That is, Ras, in coordination with membrane phosphoinositides, recruits Grb14 to the cell membrane in close proximity to the insulin receptor, facilitating the interaction of BPS and SH2 domains with the kinase domain, resulting in its inhibition.The interaction with Ras, for which we have biochemical data, is specific to Grb14 and Grb7. The other family member, Grb10, does not bind well to Ras despite ~50% sequence identity in the RA domain. Crystallographic studies of Grb14 RA-PH in complex with GTP-bound (activated) Ras, for which we seek mail-in synchrotron data collection, will shed light on the mode of binding between these two proteins and provide the structural basis for the recruitment of Grb14 by activated Ras.